RU2501855C2 - Cleaning compositions containing transglycosidase - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention proposes use of a composition containing transglycosidase ferment (EC 2.4.1.24) for decomposition of polysaccharide of natural gum. The above polysaccharide of natural gum is a substrate for the above ferment of transglycosidase. A natural gum destruction method is described, which involves contact of transglycosidase ferment with polysaccharide of natural gum for destruction of the above polysaccharide of natural gum. Besides, a cleaning method is proposed, which involves contact of an object contaminated with polysaccharide of natural gum with cleaning composition including transglycosidase ferment; and maintenance of the above object and cleaning composition under conditions sufficient for effective destruction of polysaccharide of natural gum, and thus, cleaning of the above object.

EFFECT: invention allows decomposing natural gums by means of transglycosidase ferment.

19 cl, 7 dwg, 4 ex

Description

FIELD OF THE INVENTION

The present invention relates to compositions containing the transglucosidase enzyme and a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also relates to methods of using the transglucosidase enzyme to break down the natural gum polysaccharide. In some preferred embodiments, the implementation of these compositions and methods find use in cleaning.

Prior art

Detergent and other cleaning compositions often include a complex combination of active ingredients. For example, many cleaning products contain a surfactant system, cleaning enzymes, bleaching agents, detergents, foaming suppressants, dirt suspending agents, dirt releasing agents, fluorescent brighteners, emollients, dispersing agents, dye transfer inhibiting compounds, abrasive agents, bactericidal agents and perfumes. However, despite the complexity of modern detergents, there are many contaminants that are difficult to remove. Thus, in the art there remains a need for compositions and methods for efficiently cleaning various contaminants.

Summary of the invention

The present invention relates to compositions containing the transglucosidase enzyme and a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also relates to methods of using the transglucosidase enzyme to break down the natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning.

In some embodiments, the compositions include natural gum polysaccharides, such as xanthan gums. In some additional embodiments, the transglucosidase enzyme has an activity defined as EC 2.4.1.24 according to the IUBMB nomenclature. In some further embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to the Aspergillus transglucosidase enzyme. In some other embodiments, the composition further comprises at least one surfactant. In some further embodiments, the natural gum polysaccharide is present on the subject as contamination. In some particularly preferred embodiments, the subject is tissue.

The present invention also provides methods comprising combining a transglucosidase enzyme with at least one natural gum polysaccharide to break down a natural gum polysaccharide. In some embodiments, the transglucosidase enzyme has an activity defined as EC 2.4.1.24 according to the IUBMB nomenclature. In some embodiments, the natural gum polysaccharide is xanthan gum.

The present invention also relates to cleaning methods, including: contacting an object contaminated with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and maintaining the subject and the cleaning composition under conditions sufficient to effectively destroy the polysaccharide of natural gum and thereby clean the subject. In some embodiments, the subject is contaminated with food that contains at least one natural gum polysaccharide. In some further embodiments, the natural gum polysaccharide comprises xanthan gum. In some other embodiments, the subject is selected from fabrics and hard coatings. In some further embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to the Aspergillus transglucosidase enzyme. In some embodiments, the implementation of the cleaning composition further includes at least one surfactant. In some additional embodiments, the implementation of the cleaning composition further includes at least one enzyme selected from proteases, amylases, cellulases, lipases, cutinases, mannanases, pectinases, pectatliases and oxidoreductases to destroy other contaminants. In some other additional embodiments, the implementation of the cleaning composition has a pH from about pH 5.0 to approximately pH 11.5. In some other additional embodiments, the implementation of the cleaning composition includes a transglucosidase enzyme in a concentration in the range of from about 0.01 ppm to about 100 ppm.

In some embodiments, the present invention relates to compositions comprising: a transglucosidase enzyme; and at least one natural gum polysaccharide; where the natural gum polysaccharide is a substrate for the transglucosidase enzyme. In some embodiments, a natural gum polysaccharide comprises xanthan and / or guar gum. However, it is not intended that the present invention be limited to any particular gum polysaccharide. In some preferred embodiments, the natural gum polysaccharide is present as a fouling on an object (e.g., tissue), while in other embodiments, it is in a free state in solution. In some embodiments, the transglucosidase enzyme comprises an amino acid sequence that is at least about 70% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least approximately 95% identical or at least approximately 98% identical to Aspergillus transglucosidase. In some embodiments, the compositions further comprise at least one surfactant and / or other cleaning agent.

The present invention also relates to methods that include combining a transglucosidase enzyme with at least one natural gum polysaccharide to break down a natural gum polysaccharide. In some embodiments, the methods include: contacting an object contaminated with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and maintaining the subject and the cleaning composition together under conditions sufficient to effectively destroy the polysaccharide of natural gum and thereby clean the subject.

In some embodiments, an object (eg, tissue) is contaminated with food that contains at least one natural gum polysaccharide (eg, food that contains at least one xanthan and / or guar gum). In some embodiments, the cleaning composition further comprises at least one surfactant, other cleaning agents, and / or at least one additional enzyme (eg, protease, amylase, cellulase, lipase, cutinase, oxidoreductase, or the like) to destroy others polluting components. In some embodiments, the cleaning compositions have a pH in the range of from about pH 5.0 to about pH 11.5, and the transglucosidase enzyme is present in the cleaning composition in a concentration of from about 0.01 ppm to about m.

In some embodiments, the methods provided herein lead to more efficient removal of contaminants that contain natural gum polysaccharides than equivalent methods that do not use the transglucosidase enzyme.

In some further embodiments, the present invention provides an isolated enzyme comprising Aspergillus transglucosidase produced by the Trichoderma reesei host cell. The present invention also relates to cleaning compositions (for example, detergents for washing) containing this enzyme, as well as to methods of using this enzyme to clean an object (for example, fabric).

Description of drawings

Some aspects of the following detailed description are best understood when read in conjunction with the accompanying figures. It is emphasized that, in accordance with general practice, the various details of the figures are not given to scale. On the contrary, the dimensions of the various parts are conventionally increased or reduced for clarity.

The figure 1 presents a map of the vector pTrex3 (AGL51M).

Figure 2 shows the nucleotide sequence of the expression cassette pTrex3 (AGL51M) (SEQ ID NO: 1).

The figure 3 presents a histogram showing the enzymatic activity of transglucosidase at 0.2% xanthan gum when measured using a sugar recovery test.

Figure 4 shows histograms showing the effectiveness of cleaning Trip-TG in 50 mM Hepes buffer (pH 7.4) and in AATCC HDL detergent (pH 7.4) on guar contaminated microsamples (top panel) and salad samples contaminated with salad dressing (bottom) panel).

The figure 5 presents a histogram of the experiment on the dose-response response, demonstrating that Trip-TG is active on contaminated salad dressing microsamples in a concentration of from 1 to 5 ppm in AATCC HDL.

Figure 6 is a bar graph showing the cleaning activity of Trip-TG on salad dressing contaminated microsamples in a heavy duty solid detergent (HDD).

7 is a bar graph showing the cleaning activity of Trip-TG on jam contamination in the Tergotometer test using 0.15% AATCC HDL.

Description of the invention

The present invention relates to compositions containing the transglucosidase enzyme and a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also relates to methods of using the transglucosidase enzyme to break down the natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning.

Before experimental embodiments will be described in more detail, it should be understood that this invention is not limited to the particular described embodiments, and as such may, of course, vary. It should also be understood that the terminology used in the present description is intended to describe only specific embodiments and is not considered to be limiting.

When used in the present description, when a range of values is proposed, it should be understood that each intermediate value is up to a tenth of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of this range is also specifically disclosed. Each smaller range between any set value or an intermediate value in a set range and any other set or intermediate value in this set range is encompassed by the invention. The upper and lower limits of these smaller ranges in the range can be independently included in or excluded from the range, and each range, where one or the other, neither one or the other, or both limits are included in smaller ranges, is also covered by the invention depending on any specifically excluded limit within the specified range. When the established range includes one or both limits, ranges excluding one or the other, or both of these included limits, are also included in the invention.

Although any methods and materials similar or equivalent to those described herein can be used in the methods or testing of the present invention, illustrative methods and materials will be described below. All patents and publications cited herein are incorporated herein by reference for disclosing and describing methods and / or materials, including the sequences in which publications are cited. Any entry numbers in the GENBANK® database listed herein are incorporated by reference in their entirety, including the nucleic acid and protein sequences indicated therein and comments on these sequences starting from the earliest filing date of this patent application.

The publications discussed herein are offered solely because of their disclosure prior to the filing date of this application. Nothing in the present description should be construed as the assumption that the present invention does not give the right to retroactively date such a publication on the basis of the previous invention. Further, the dates of the publication provided may differ from the actual publication dates, which may need independent confirmation.

Unless otherwise indicated herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although many methods and materials similar or equivalent to those described herein find use in the methods or testing of the present invention, some of the preferred methods and materials are described herein. Digital ranges include numbers that specify the range, as well as each digit in between.

Definitions

When used in the present description and the attached claims, the singular includes a reference to the plural, unless the context clearly indicates otherwise. Thus, for example, reference to a “host cell” includes a plurality of such host cells.

Unless otherwise indicated, nucleic acids are presented from left to right in the orientation from the 5'- to 3'-end; amino acid sequences are presented from left to right in the orientation from amino to carboxyl end, respectively. The chapters provided herein do not limit the various aspects of the embodiments of the invention that can be obtained by reference to the description as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the description as a whole.

The term “recombinant” means a polynucleotide or polypeptide that does not naturally exist in the host cell. In some embodiments, the recombinant molecules comprise two or more naturally occurring sequences that are linked together in a manner that does not exist in nature. A recombinant cell contains a recombinant polynucleotide or polypeptide.

The term “heterologous” refers to elements that are normally not related to each other. For example, if a host cell produces a heterologous protein, this protein is normally not produced in this host cell. In the same way, a promoter that is operably linked to a heterologous coding sequence is a promoter that is operably linked to a coding sequence with which it is not normally operably linked in a wild-type host cell. The term “homologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that exists in a host cell in nature.

The terms “protein” and “polypeptide” are used interchangeably herein.

A “signal sequence” is an amino acid sequence present on the N-terminal portion of a protein that facilitates the secretion of the mature form of the protein from the cell. Signal sequence determination is functional. The mature form of extracellular protein lacks a signal sequence that is cleaved during secretion.

A “coding sequence” is a segment of DNA that encodes a polypeptide.

The term "nucleic acid" includes DNA, RNA, single-stranded or double-stranded, and their chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein.

“Vector” means a polynucleotide designed to introduce nucleic acids into one or more host cells. Suitable vectors autonomously replicate in various host cells and include: cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

As used herein, the term “expression vector” refers to a DNA construct comprising a protein coding region that is operably linked to a suitable control sequence capable of influencing protein expression in a suitable host cell. In some embodiments, such control sequences include a transcriptional promoter, an optional transcription control operator for mRNA production, a sequence encoding suitable ribosome binding sites for mRNA, and enhancers and other sequences that control transcription and translation termination.

A “promoter" is a regulatory sequence that initiates transcription of an underlying nucleic acid.

The term "operatively linked" means the location of the elements, which allows them to be functionally connected. For example, a promoter is operably linked to a coding sequence if it controls transcription of the sequence.

The term "selective marker" refers to a protein capable of expression in the host, which makes it easy to select those hosts that contain the introduced nucleic acid or vector. Examples of selective markers include, but are not limited to, antibiotic resistance genes (e.g. hygromycin, bleomycin or chloramphenicol) and / or genes that confer a metabolic advantage, such as an advantage in the nutrition of the host cell.

The term “originating” encompasses the terms “originating from”, “obtained”, “obtained from” and “isolated from” and refers to a source of sequence and / or protein.

A “non-pathogenic” organism is an organism that is not pathogenic to humans and / or other animals.

As used herein, the terms “isolate,” “isolate,” and “separate” refer to a protein, cell, nucleic acid, or amino acid that is isolated from at least one component with which they are naturally associated.

Used in the present description, the terms "transformed" and "transgenic" used in relation to a cell, mean that the cell has a non-natural (eg heterologous) nucleic acid sequence integrated in its genome or in the form of an episomal plasmid, the existence of which has been maintained for many generations.

As used herein, the term “expression” refers to a process by which a polypeptide is produced based on a nucleic acid sequence of a gene. The process includes both transcription and translation.

The term “introduced” in the context of introducing a nucleic acid sequence into a cell refers to “transfection”, “transformation” or “transduction” and includes reference to the introduction of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid sequence can be introduced into the genome of the cell ( for example, to a chromosome, plasmid, plastid or mitochondrial DNA), converted to an autonomous replicon or temporarily expressed (for example, transfected mRNA).

The term “hybridization” refers to a process by which a nucleic acid strand is joined to a complementary strand by forming base pairs, as is known in the art. A nucleic acid is considered to be “selectively hybridizable” with a reference nucleic acid sequence if the two sequences specifically hybridize to one another under conditions of moderate to high stringency of hybridization and washing. Conditions of moderate and high stringency hybridization are known to specialists in this field of technology. One example of high stringency conditions involves hybridization at about 42 ° C in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg / ml denatured DNA carrier, followed by washing twice in 2X SSC and 0.5 % SDS at room temperature and an additional two times 0.1 X SSC and 0.5% SDS at 42 ° C.

As used herein, the terms “cleaning composition” and “cleaning composition” refer to a composition that finds use in removing unwanted compounds (eg, “contaminants”) from objects to be cleaned, such as fabrics, dishes, contact lenses, other hard substrates, hair (shampoos), skin (soap and creams), teeth (mouthwashes and toothpastes). The term encompasses any materials / compounds selected for a particular type of cleaning composition in the desired product form (e.g., liquid, gel, granules or spray composition), provided that the composition is compatible with the test enzyme in the composition. A specific selection of materials of the cleaning composition is easily carried out when considering the surface, objects and / or fabric being cleaned, and the desired shape of the composition when used for cleaning conditions.

The terms are intended to include, but are not limited to, detergent compositions (e.g., liquid detergents, gel detergents and / or solid detergents for washing and detergents for delicate fabrics; cleaning compositions for hard surfaces such as glass, wood, ceramic and metal surfaces of the counter and windows; cleaning compositions for carpets; cleaning compositions for ovens; fabric fresheners; fabric softeners; and preliminary stain removers for textiles and laundry, as well as detergents for dishes).

As used herein, the term “cleaning composition” includes, unless otherwise indicated, granular, tablet detergents or detergents in powder form for all purposes, or heavy duty detergents, especially cleaning detergents; detergents in the form of a liquid, gel or in the form of a paste for all purposes, especially the so-called heavy-duty liquid types (HDL); liquid detergents for thin tissues; manual dishwashing agents or light dishwashing agents, including the type of high foaming agents; detergents for the dishwasher, including various tableted, granular, liquid types and types of rinses for the absence of drops on the dishes for use at home and in institutions; liquid cleaning and disinfecting agents, including antibacterial types for washing by hand, cleaning bars, mouthwashes, cleaning agents for dentures, shampoos for machines or carpets, cleaning agents for bathrooms; hair shampoos and hair rinses; shower gels and bath foams; cleaning agents for metal; as well as cleaning auxiliaries, such as bleaching and stain stickers, types of pretreatment or laundry additives.

As used herein, the terms “detergent composition” and “detergent composition” are used to refer to compositions that are formulated for use in a cleaning medium to clean contaminated items. In specific embodiments, the term is used to refer to washable fabrics and / or garments (eg, “laundry detergents”). In alternative embodiments, the term refers to other detergents, such as detergents used for washing dishes, cutlery, etc. (for example, “dishwashing detergents”). It is not intended that the present invention be limited to any particular detergent composition or composition. Indeed, it is understood that, in addition to the test enzyme, the detergent composition may also include surfactants, transferase (s), hydrolytic enzymes, oxidoreductases, detergent components, bleaching agents, bleaching activators, blueing agents and fluorescent dyes, caking inhibitors, masking agents enzyme activators, antioxidants and / or solubilizers, etc.

As used herein, the term “enhanced effectiveness” in a cleaning composition is defined as enhanced cleaning (eg, removal and / or discoloration) of stains (especially stains including natural gum polysaccharides such as chocolate cream, salad dressing, guar, etc.) when determined using a normal assessment after a standard wash cycle.

As used herein, the term “hard surface cleaning composition” refers to detergent compositions for cleaning hard surfaces such as floors, walls, tiles, bathtubs and kitchen appliances and the like. Such compositions are offered in any form, including, but not limited to, solid agents, liquids, emulsions, etc.

As used herein, the term “dishwashing composition” refers to all forms of dishwashing compositions, including, but not limited to, gel forms, granular and liquid forms.

As used herein, the term “fabric cleaning composition” refers to all forms of detergent fabric cleaning compositions, including, but not limited to, gel form, granular, liquid form and bar form.

Used in the present description, the term "textile" refers to textile fabrics, as well as staple fibers and threads suitable for turning into yarn or for use as yarn, textile, knitted and non-textile fabrics. The term covers yarn made from natural as well as synthetic (e.g. processed) fibers.

Used in the present description, the term "fabric" covers any textile material. Thus, the term is intended to encompass clothing items, as well as fabrics, yarn, fibers, non-textile materials, natural materials, synthetic materials, and any other textile material.

As used herein, the term “effective amount of transglucosidase” refers to the amount of transglucosidase enzyme required to achieve the enzymatic activity required for a particular application (eg, in a cleaning composition, etc.). Such effective amounts are readily established by one skilled in the art and are based on many factors, such as the particular enzyme variant used, the use of cleansing, the specific composition of the cleaning composition, and the need for a liquid or dry (e.g., granular, block) composition and the like.

The term "transglucosidase" refers to an enzyme that transfers the residue of D-glucosyl in 1,4-α-D-glucan to the primary hydroxyl group of glucose, free or connected to 1,4-α-D-glucan. The transglucosidase described herein has the activity described as EC 2.4.1.24 in accordance with the IUBMB enzyme nomenclature. The systematic name of the transglucosidase described herein is 1,4-α-D-glucan: 1,4-α-D-glucan (D-glucose) -6-α-D-glucosyl transferase. This enzyme may be referred to in some publications as α-glucosidase.

The term “contaminated item” refers to an item (eg, cloth or utensil) that is contaminated (eg, “tinted”) by the second composition. The term “contaminated item” encompasses dirty fabrics such as dirty clothes, linens, and fabrics dyed with foods containing natural gum polysaccharide. In some embodiments, when stained, visible staining occurs, while in other embodiments, visible staining does not appear.

The term "natural gum polysaccharide" refers to a naturally occurring polysaccharide that is not starch, which is capable of causing a significant increase in solution viscosity at low concentration. Such polysaccharides are commonly used in the food industry and are used as thickeners, gelling agents, emulsifiers and stabilizers in many food products (e.g., sauces, creams, dairy products, ice cream, mousses, milkshakes, salad dressings, etc.). Guar gum (food supplement E412), an edible thickener extracted from the legume of guar bushes, and xanthan gum (food supplement E415), a polysaccharide that is produced by fermenting glucose or sucrose (for example, using Xanthomonas campestris), are examples of . Other natural gum polysaccharides include, but are not limited to, agar (E406), alginic acid (E400), β-glucan, carrageenin (E407), natural rubber gum, dammar gum, gellan gum (E418), glucomannan (E425), gum (E414), ghati gum, tragacanth gum (E413), karaya gum (E416), robinia bean gum (E410), pistachio gum, sodium alginate (E401), spruce gum and container gum (E417).

The term “non-starch food polysaccharide-degrading enzyme” means an enzyme that destroys non-starch food polysaccharides. Examples of enzymes include, but are not limited to, hemicellulase, mannase, pectinase, xylanase, β-galactosidase and α-galactosidase.

The term "working pH" refers to the pH of the detergent during its use. For example, the working pH of the detergent for washing is the pH of the detergent when it is used to wash fabrics in a washing machine and / or when washing clothes manually. Similarly, the working pH of the dishwashing detergent is the pH of the detergent when it is used in the dishwasher and / or when washing dishes manually. In some embodiments, detergents that are in concentrated or solid form are diluted or dissolved before the pH of the detergent becomes its working pH.

The term "working concentration" refers to the concentration of the enzyme in the detergent during its use. For example, the working concentration of the enzyme in the detergent for washing is the concentration of this enzyme when using detergent for washing in the process of washing fabrics in a washing machine and / or when washing clothes manually. Similarly, the working concentration of an enzyme in a dishwashing detergent is the concentration of this enzyme in a detergent when it is used in a dishwasher and / or when washing dishes manually. In some embodiments, detergents that are in concentrated or solid form are diluted or dissolved before the concentration of the enzyme in the detergent becomes its working concentration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions containing the transglucosidase enzyme and a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also relates to methods of using the transglucosidase enzyme to break down the natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in purification. Indeed, the present invention relates to various compositions containing transglucosidase enzymes, as well as to methods of using the compositions.

Transglucosidase Enzyme Containing Compositions

As noted above, the present invention relates to compositions containing transglucosidase. In some embodiments, the composition comprises at least one transglucosidase enzyme and a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme.

As noted above, the transglucosidase enzyme usually has an activity defined as EC 2.4.1.24 according to the IUBMB enzyme nomenclature, this activity transferring glycosyl residues in certain glycans to the primary hydroxyl group of glucose. In some embodiments, the enzyme may also have an activity that breaks down the natural gum polysaccharide (e.g., xanthan and polysaccharides containing galactomannan, such as guar gum or lima bean gum) by cutting the sugar side chains or breaking internal bonds to break down the polysaccharide backbone.

Any suitable transglucosidase enzyme finds use in the present invention (see, for example, Pazur et al., Carbohydr. Res. 1986 149: 137-47 [1986]; and Nakamura et al., J. Biotechnol., 53: 75-84 [1997]). In some embodiments, transglucosidase enzymes useful in the present invention are commercially available (for example, but not limited to, enzymes supplied by Megazyme, Wicklow, Ireland; or Danisco US Inc., Genencor Division, Palo Alto, CA). In some embodiments, the enzyme is an Aspergillus niger transglucosidase produced by Trichoderma reesei cells. In some further embodiments, the transglucosidase is a wild-type fungal transglucosidase (e.g., but not limited to, a fungal transglucosidase having the amino acid sequence deposited in the NCBI's GENBANK® database with registration numbers: D45356 (GID: 2645159; Aspergillus niger), BAD06006 (GID: 4031328; Aspergillus awamori), BAA08125.1 (GID: 1054565; Aspergillus oryzae), XP_001210809.1 (GID: 115492363; Aspergillus terreus), XP_001271891.1 (GID: 121707620; Aspergillus clavatus) : 119500484; Neosartorya fischeri), XP_751811.1 (GID: 70993928; Aspergillus fumigatus), XP_659621.1 (GID: 67523121; Aspergillus nidulans), XP_001216899.1 (GID: 115433524; Aspergillus terreus) XP_001258585.1 (GID: 119473371; Neosartorya fischeri)), or a variant thereof, which has an amino acid sequence that is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical wild-type fungal transglucosidase.

In some preferred embodiments, the enzyme is typically present in the composition in a concentration range from about 0.01 ppm (ppm, w / v) to about 100 ppm (for example, from about 0.01 ppm to about 0 05 ppm, from about 0.05 ppm to about 0.1 ppm, from about 0.1 ppm to about 0.5 ppm, from about 0.5 ppm to about 1 ppm, from about 1 ppm to about 5 ppm, from about 5 ppm to about 10 ppm, from about 10 ppm to about 100 ppm).

In some preferred embodiments, the composition is a cleaning composition. In some particularly preferred embodiments, the composition further comprises at least one surfactant, cleaning agents, and / or other tissue care agents, described in more detail below.

In some embodiments, the natural gum polysaccharide is in aqueous solution, while in other embodiments, it is adhered to an object (for example, as a spot that is on the surface of an object, such as a fabric or a hard surface). In some embodiments, the natural gum polysaccharide is in dried form. Since natural gum polysaccharides are widely used in a variety of food products, in some embodiments, the subject is contaminated with a food product that contains a natural gum polysaccharide. Typical foods that contain a natural gum polysaccharide include, but are not limited to, sauces, creams, dairy products, ice cream, mousses, milkshakes, salad sauces, fruit juice drinks, canned fruit, jellies, soy sauce, oyster sauce, pre-packaged foods, cheese and bakery products. In some embodiments, the natural gum polysaccharide is in the food product at a concentration of from about 0.1% to about 1.5%, from about 0.1% to about 0.5%, from about 0.5% to about 1.0 %, approximately 1.0% or approximately 1.5%.

In some preferred embodiments, transglucosidase is prepared using conventional methods. For example, in some embodiments, it is secreted into the periplasm (e.g., gram-negative organisms such as E. coli) or into the extracellular space (e.g. gram-positive organisms such as Bacillus and Actinomycetes) or eukaryotic hosts (e.g. Trichoderma, Aspergillus, Saccharomyces and Pichia).

In some embodiments, a transglucosidase enzyme is obtained by expressing a fusion protein containing a signal sequence operably linked to a transglucosidase enzyme in a T. reesei host cell. In some of these embodiments, the transglucosidase enzyme is secreted into the culture medium from which it is recovered. Any signal sequence that facilitates the secretion of a protein from a Trichoderma host cell is used in suitable fusion proteins. In some embodiments, the signal sequence is endogenous, while in other embodiments, it is not endogenous to the Trichoderma host cell, and in some embodiments, it is the signal sequence of a protein that is known to be secreted from the Trichoderma sp host cell . at a high level. Such a signal sequence includes, but is not limited to: the signal sequence of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanase III, α-amylase, aspartyl proteases, glucoamylase, mannanase, glycosidase and endopeptidase B of barley, e.g. Appl. Environ. Microbiol. 63: 4938-4940 [1997]). In some embodiments, implementation, and as further described in the Examples section of the present disclosure, a transglucosidase is secreted using its own signal sequence (i.e., AGL1, AGL2 or AGL3 signal sequences).

In some embodiments, implementation, therefore, transglucosidase is obtained using a nucleic acid, which includes: a nucleic acid encoding a signal sequence operably linked to a nucleic acid encoding a transglucosidase, where the translation of the nucleic acid gives a fusion protein comprising a transglucosidase portion containing an N-terminal signal a sequence for secretion of the transglucosidase moiety from a Trichoderma host cell.

In some embodiments, the fusion protein, in addition to the signal sequence, further comprises a “carrier protein”, which is a portion of the protein that is endogenous and secreted by the T. reesei sp. at a high level. Suitable carrier proteins include, but are not limited to, T. reesei mannanase I proteins (Man5A or MANI), T. reesei cellobiohydrolases II (Cel6A or CBHII) (see, for example, Paloheimo et al., Appl. Environ. Microbiol. 69: 7073-7082 [2003]) or cellobiohydrolase I T. reesei (CBHI). In some embodiments, the carrier protein is a truncated T. reesei CBH1 protein that includes a central region of CBH1 and a portion of the linker region of CBH1. Thus, in some embodiments, a nucleic acid is used that encodes a protein containing a signal sequence, a carrier protein and the phytase in question, functionally linked from the amino terminus to the carboxyl end.

In some embodiments, the coding sequence of the transglucosidase is codon-optimized for expression of the transglucosidase in the host cell used. Because codon usage tables are known or can be readily obtained in the art that show the use of each of the codons in many host cells, including Trichoderma reesei (see, for example, Nakamura et al., Nucl. Acids Res., 28: 292 [2000]), such nucleic acids can be easily constructed taking into account the amino acid sequence of the transglucosidase to be expressed.

In some embodiments, the nucleic acid, in addition to the coding sequence, further comprises other elements that are necessary for the expression of the transglucosidase enzyme in the host cell. For example, in some embodiments, the nucleic acid comprises a promoter for transcribing a coding sequence and a transcription terminator. Typical promoters that find use in T. reesei include, but are not limited to, the cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 promoters of T. reesei, or a hybrid or truncated variant thereof. For example, in some embodiments, the promoter is a T. reesei cbh1 promoter. Suitable terminators include the terminators cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 T. reesei and many others, including, for example, terminators of the glucoamylase genes A. niger or A. awamori, as is known to those skilled in the art, as well as genes Aspergillus nidulans antranylate synthases, Aspergillus oryzae TAKA amylase genes or A. nidulans trpC (Punt et al., Gene 56: 117-124 [1987]). In some embodiments, the promoter and / or terminator is natural for the Trichoderma sp. Host cell, while in other embodiments, they are not endogenous for the Trichoderma sp. Host cell.

In some embodiments wherein T. reesei is used as a host cell to express the transglucosidase enzyme, the cell is genetically modified to reduce the expression of secreted proteins endogenous to the cell. In some embodiments, the implementation of the cell includes one or more natural genes, in particular genes that encode secreted proteins that have been removed or inactivated. For example, in some embodiments, one or more protease encoding genes is deleted or inactivated (eg, an aspartyl protease encoding gene; see Berka et al., Gene 86: 153-162 [1990]; and US Patent No. 6509171), or genes encoding cellulase. In some embodiments, the Trichoderma sp. is a T. reesei host cell containing inactivating deletions in the cbh1, cbh2, egl1 and egl2 genes, as described in patent WO 05/001036. In some embodiments, the nucleic acid described above is located in the genome of a Trichoderma sp. Host cell, while in other embodiments, it is in a plasmid that replicates in a Trichoderma host cell.

Any method for introducing nucleic acids into Trichoderma host cells can be used in the present invention (for example, electroporation, nuclear microinjection, transduction, transfection, transfection mediated by lipofection and DEAE-dextrin, incubation with precipitate of DNA with calcium phosphate, high-speed microbial bombardment coated with DNA protoplast fusion). Indeed, conventional transformation methods are well known in the art (see, for example, Campbell et al., Curr. Genet., 16: 53-56 [1989]; patent WO 05/001036; US patent No. 6022725; US patent No. 6103490; US patent No. 6268328; and published US patent applications Ser. No. 20060041113, 20060040353, 20060040353 and 20050208623, and these publications are incorporated into this description by reference). In some embodiments, preparing Trichoderma for transformation involves preparing protoplasts from the mycelium of the fungus. (See, for example, Campbell et al., Above). In some embodiments, the mycelium is obtained from sprouted vegetative spores.

In some embodiments, the transglucosidase enzyme, after secretion into the culture medium, is recovered using any suitable conventional method (for example, by precipitation, centrifugation, affinity, filtration, or any other method known in the art. For example, affinity chromatography is used (Tilbeurgh et al., FEBS Lett., 16: 215 [1984]); ion exchange chromatography methods (Goyal et al., Biores. Technol., 36:37 [1991]; Fliess et al., Eur. J. Appl. Microbiol., Biotechnol., 17: 314 [1983]; Bhikhabhai et al., J. Appl. Biochem., 6: 336 [1984]; and Ellouz et al., Chromatogr., 396: 307 [1987]), including ionic high-resolution substances (Medve et al., J. Chromatogr. A 808: 153 [1998]; chromatography with hydrophobic interaction (Tomaz and Queiroz, J. Chromatogr. A 865: 123 [1999]; two-phase distribution ( Brumbauer et al., Bioseparation 7: 287 [1999]); ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or gel filtration (for example, using Sephadex G-75). In some embodiments, transglucosidase is used without purification from other components of the culture medium. In some embodiments, the culture medium is simply concentrated and then used without further purification of the protein from the components of the growth medium, while in other embodiments, it is used without any further modification or processing.

The present invention also provides methods for breaking down natural gum polysaccharides. In some embodiments, the methods include combining (eg, mixing) the transglucosidase enzyme with a natural gum polysaccharide under such conditions that the natural gum polysaccharide is destroyed. Conditions suitable for transglucosidase enzyme activity (e.g., temperature range, pH range, and other reaction components suitable for transglucosidase enzyme activity) are known in the art.

Cleaning methods

In addition to the compositions described above containing transglucosidase, the present invention also provides purification methods. These methods typically include: contacting an object contaminated with at least one natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and joint holding of the subject and the cleaning composition under conditions sufficient to effect the destruction of the polysaccharide of natural gum and thereby cleaning the subject.

In some preferred embodiments, the cleaning composition used in these methods is a fabric cleaning composition (e.g., a detergent), a surface cleaning composition, a dishwashing composition, a surfactant composition for an automatic dishwasher, and the like . Various compositions of cleaning compositions find use in the present invention, including but not limited to those described in detail in WO 0001826, incorporated herein by reference.

In some embodiments, the implementation of the composition for cleaning an item (for example, a surfactant for washing) includes from about 1% to about 80% (for example, from about 5% to about 50% (by weight)) of at least one surfactant substances (e.g., a nonionic surfactant, a cationic surfactant, an anionic surfactant and / or an amphionic surfactant, or a mixture thereof, such as a mixture of an anionic and nonionic surfactant sto-active substances). Typical surfactants include, but are not limited to: alkyl benzene sulfonate (ABS), including linear alkyl benzene sulfonate and linear sodium alkyl sulfonate, alkyl phenoxypolyethoxyethanol (e.g. nonylphenoxyethoxylate or nonylphenol), diethanolamine, triethanolamine and. Typical surfactants that are used in various cleaning products, especially laundry detergents, are described in US Pat. Nos. 3,664,961, 3,919,678, 4,222,905 and 4,239,659, each of which is incorporated herein by reference.

In some embodiments, the composition for cleaning an item is a solid (eg, in the form of a powder or tablet), liquid or gel. In some preferred embodiments, the composition further comprises at least one buffer (eg, sodium carbonate or sodium bicarbonate), a major detergent, bleach, bleach activator, enzyme, stabilizing enzyme, foam enhancer, quencher, anti-resins, anticorrosive agent mud suspending agent, mud releasing agent, bactericidal agent, pH adjusting agent, alkalizing source other than the main detergent, chelating agent, organic or inorganic filler, solvent, hydrotroph, fluorescent brightener, dye and / or perfume. In some embodiments, the implementation of the cleaning composition is combined with a detergent before use as an additive for washing.

In some embodiments, the cleaning compositions further comprise at least one enzyme that disrupts a non-starch food polysaccharide (e.g., hemicellulase, mannanase, pectinase, xylanase or pectatliase), and optionally one or more other enzymes (e.g., proteases, such as subtilisin protease and / or SSI protein; lipase, amylase, cellulase, cutinase, lipase, oxidoreductase, etc.) to remove other contaminants.

The compositions provided herein use many other ingredients suitable for detergent cleaning compositions, including but not limited to other active ingredients, carriers, hydrotrophs, processing aids, colorants or pigments, solvents for liquid formulations, etc. In those embodiments in which an additional contribution of soap is desired, foam enhancers such as C ₁₀ -C ₁₆ alkolamides, typically from about 1% to about 10% by weight of the composition, are included in the composition.

In some embodiments, detergent compositions include water and other solvents as carriers. As such carriers, low molecular weight primary or secondary alcohols such as methanol, ethanol, propanol and isopropanol are used. In some embodiments, monohydric alcohols are preferred for dissolving surfactants, but polyols, such as polyols containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxyl groups (e.g., 1,3-propanediol, are also used). , ethylene glycol, glycerin and 1,2-propanediol). In some further embodiments, the compositions comprise from about 5% to about 90%, usually from about 10% to about 50% of such carriers.

In some preferred embodiments, the detergent compositions provided herein are formulated such that during use in aqueous purification procedures, the wash water has a pH between about 5.0 and about 11.5. Thus, final products are usually made up in this range. Recommended pH control methods include buffers, alkalis, acids, etc. and are well known to those skilled in the art. In some embodiments, the implementation of the cleaning compositions are detergents for automatic dishwashers having a working pH range from about pH 9.0 to about pH 11.5, from about pH 9.0 to about pH 9.5, from about pH 9.5 to about pH 10.0, from about pH 10.0 to about pH 10.5, from about pH 10.5 to about pH 11.0, or from about pH 11.0 to about pH 11.5. In some other embodiments, the implementation of the cleaning compositions are liquid detergents for washing, having a working pH range from approximately pH 7.5 to approximately pH 8.5, from approximately pH 7.5 to approximately pH 8.0, or from approximately pH 8.0 to approximately pH 8.5. In some other embodiments, the implementation of the cleaning compositions are solid laundry detergents having a working pH range from about pH 9.5 to about pH 10.5, from about pH 9.5 to about pH 10.0, or from about pH 10.0 to approximately pH 10.5.

Various whitening compounds, such as percarbonates, perborates and the like, are typically used in the various compositions provided herein, typically at a level of from about 1% to about 15% by weight. If desired, the compositions may also contain bleach activators such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonate and the like, which are also known in the art. The levels used typically range from about 1% to about 10% by weight.

Various dirt-removing agents are also used in various compositions, especially agents such as anionic oligoesters, various chelating agents, especially aminophosphonates and ethylene diamino disuccinates, various clay-removing agents, especially ethoxylated tetraethylene pentanamine, various dispersing agents, especially polyacrylates, polyol and acrylates especially anionic brighteners, various foam absorbers, especially silicones and secondary alcohols, various fabric softeners, especially bentonite clay, at a level in the range of from about 1% to about 35% by weight. Standard formulations and published patents contain many detailed descriptions of such conventional substances that find use in the compositions of the present invention.

In some embodiments, enzyme stabilizers also find use in the compositions provided herein. Such stabilizers include, but are not limited to, propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about one%).

In some embodiments, hard surface cleaning compositions and / or fabric cleaning compositions are comprised of various major detergents from about 5% to about 50% by weight. Typical base detergents include, but are not limited to, 1-10 micron zeolites, polycarboxylates such as citrate and oxidized disuccinates, layered silicates, phosphates and the like. Other conventional base detergents are provided in standard formulations and find use in the present invention.

Other optional ingredients include chelating agents, clay mud removers / anti-redeposition agents, polymeric dispersing agents, bleaches, brighteners, foam suppressants, solvents and aesthetic agents.

The cleaning methods of the present invention are more effective in removing certain stains (for example, stains from foods containing natural gum polysaccharides) compared to equivalent methods that do not use the transglucosidase enzyme. In some preferred embodiments, compared to an otherwise equivalent method that does not contain the transglucosidase enzyme, the cleaning compositions of the present invention are more effective in stain removal. When using a standard OTDR test, for example, the method in question provides removal and / or discoloration of at least about 20%, at least about 40%, at least about 60%, at least about 80 %, or in some embodiments, at least about 90% more spots than the equivalent method that does not use the transglucosidase enzyme.

To further illustrate the present invention and its advantages, the following specific examples are presented with the understanding that they are offered to illustrate the present invention and should not be construed in any way as limiting its scope.

experimental part

The following examples are proposed in such a way as to provide a person skilled in the art with a full disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what applicants consider as their invention, and they are not intended to represent what the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weighted average molecular weight, temperature is presented in degrees Celsius, and pressure is equal to or close to atmospheric.

Example 1

Expression of A. niger transglucosidase in T. reesei

The nucleic acid encoding the mature A. niger transglucosidase enzyme was amplified from A. niger genomic DNA by PCR and cloned into the pTrex3 vector to obtain pTrex3 (AGL51M). pTrex3 (AGL51M) is shown in Figure 1. The transglucosidase protein encoded by this vector was operably linked to the CBH1 signal sequence to facilitate its secretion into the growth medium. The promoter and terminator T. reesei cbh1 were located next to the coding transglucosidase sequence. The nucleotide sequence of the expression cassette of the vector pTrex3 (AGL51M) is shown in figure 2.

XbaI-XbaI fragment 7.57 kb pTrex3 plasmids (AGL51M) were purified by agarose gel electrophoresis and used to transform T. reesei spores by electroporation. The electroporation parameters were as follows: voltage - 16 kV / cm, capacitance - 25 μF, resistance - 50 Ohms. Electroporation was performed using a suspension of freshly obtained T. reesei spores suspended in ice-cold 1.2 M sorbitol. After electroporation, spores were incubated overnight in a rotary shaker (30 ° C, 200 rpm) in a medium containing 1 M sorbitol, 0.3% glucose, 0.3% bactopeptone and 0.15% yeast extract. Germinating spores were plated on a selective medium containing acetamide as the sole nitrogen source (acetamide 0.6 g / l; cesium chloride 1.68 g / l; glucose 20 g / l; primary potassium hydrogen phosphate 15 g / l; magnesium sulfate heptahydrate 0.6 g / l; calcium chloride dihydrate 0.6 g / l; iron (II) sulfate 5 mg / l; zinc sulfate 1.4 mg / l; cobalt (II) chloride 1 mg / l; manganese sulfate (II ) 1.6 mg / L; agar 20 g / L; pH 4.25). Transformation colonies appeared after about 1 week. Individual transformants were transferred to fresh plates with selective acetamide and allowed to grow for 3-4 days.

Isolates exhibiting stable growth on selective medium were used to inoculate 5 ml of the described lactose medium (see, for example, patent WO 2005/001036, p. 60) in 20 × 175 mm test tubes. Tubes were fixed in a rotary shaker at an angle of approximately 45 ° and shaken at 200 rpm and 28 ° C for 4-5 days. Electrophoretic analysis of culture supernatants showed the presence of a new protein band of approximately the expected molecular weight.

The production of transglucosidase activity by transformants was also confirmed using an enzymatic test. Testing was carried out in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM para-nitrophenyl-α-glucoside and 1 mg / ml BSA. After 30 minutes of incubation at 30 ° C, the reaction was stopped by adding an equal volume of 1 M sodium carbonate and an OD of _{405 was} recorded. Typically, transformants expressed 1-2 E transglucosidase activity (expressed in micromoles of para-nitrophenol released per minute) per ml of culture broth. In non-transformed controls, activity was below the measurement limit.

Example 2

Transglucosidase Destroys Xanthan Gum

The hydrolytic activity of the enzymes against xanthan gum was measured using a sugar recovery test using the PAHBAN reagent (para-hydroxybenzoic acid hydrazide), as is known in the art (see Lever et al., Anal. Biochem., 47: 273 [1972] ) Xanthan gum (CAS 111 38-66-2) was obtained from Sigma Chemicals, St. Louis MO and dissolved in 50 mM sodium acetate pH 6.0 buffer at a concentration of 0.2%. For some experiments, AATCC standard heavy duty liquid detergent (AATCC HDL 2003 without clarifier, Test Fabrics, Inc. West Pittston, PA) was added in an amount of 1.5 ml / L (0.15%). The AATCC HDL liquid detergent contained 12% linear alkyl sulfonates, 8% alcohol ethoxylates, 8% propanediol, 1.2% citric acid, 4% fatty acid and 4% sodium hydroxide, adjusted with water.

Testing was performed on a 24-well microplate (COSTAR 3526, Corning Incorporated, Corning, NY) as follows: one ml of buffer was added to well 1, one ml of buffer plus enzyme was added to well 2, one ml of buffer and substrate was added to well 3 and well 4 was added one ml of buffer plus substrate and enzyme. For statistical purposes, each well can be repeated 2 to 4 times. Enzymes to be tested are usually diluted in a reaction buffer from 1 to 10-fold, from 1 to 1000-fold. After adding all the reagents, the microplate was covered with a plastic lid and the gap between the lid and the plate was carefully sealed with several layers of parafilm (Pechiney Plastic Packing, Menasha, WI) to prevent evaporation. The reaction plate was incubated for 1 to 16 hours at 37 ° C on a shaker rotating at 100 rpm.

Sugar-reducing activity was measured using an Eppendorf Mastercycler Gradient thermal cycler (Eppendorf Scientific, Westbury, NY) and 0.2 ml disposable tape tubes and caps for PCR (polymerase chain reaction) from VWR International, West Chester, PA. The sugar reducing reagent was prepared as follows: 0.15 g of sodium potassium tartrate tetrahydrate (Rochelle Salt, Sigma Chemical Co.) and 0.15 g of parahydroxybenzoic acid hydrazide (H-9882, Sigma) were added to 10 ml of 2% sodium hydroxide in distilled water Chemical Co.). The solution (called the "PAHBAH reagent") was rotated to dissolve all the ingredients and placed on ice in the dark before use. This reagent was re-prepared daily. Immediately prior to sample analysis, 0.160 ml of PAHBAH reagent and then 5 to 20 μl of enzyme samples and controls were added to each tube of the PCR tape. All tubes were tightly closed with lids, placed in a thermal cycler and incubated for 15 min at 99 ° C, followed by cooling to 4 ° C for at least 15 minutes. After cooling, the covers were removed from the ribbon tubes and 0.15 ml of each sample was placed in a 96-well flat-bottom microplate (COSTAR 9017, Corning Inc. Corning, NY) and read using a Spectra Max 250 plate reader (Molecular Devices, Sunnyvale, CA) at 405 nm against blank of distilled water. Each enzyme sample was analyzed as follows: the optical density (OD) of the control sample was subtracted from the OD of the sample with the buffer, and the obtained value was added to the control with the substrate and buffer. The OD of the reaction of the enzyme with the substrate was compared with the sum of the controls of the substrate and the sample.

Figure 3 shows that transglucosidases produced in Trichoderma (Trip-TG) and Aspergillus (Mega-TG) exhibit significant activity in sugar reduction on xanthan gum in 50 mM acetate buffer plus AATCC heavy-duty liquid detergent at pH 6.0.

Example 3

Transglucosidase removes contamination from contaminated flaps.

Cotton samples contaminated with pigment salad dressing (STC CFT CS-6) and guar pigment (STC CFT CS-43) were obtained from Test Fabrics, Inc. West Pittston, PA, USA. Microplate test samples were cut into 15 mm circles (discs) using a Punch Press Model B for tissues equipped with a 5/8 "die cutter. Single disc samples were placed in each well of a 24-well microplate (Costar 3526). One (1 ) ml of washing solution containing 1.5 ml of AATCC HDL detergent, 50 mM Hepes buffer and 6 to 60 ppm of the enzyme diluted in 50 mM Hepes buffer, pH 7.4, was added to each well per microplate. lid and aluminum foil and incubated at 37 ° C with careful rotation at 100 rpm in 4-16 hours. The plates were removed from the shaker and the detergent solution was removed by suction. Each well of the microplate was washed three (3) times with 1.5 ml of Dulbecco's PBS, pH 7.3, and three (3) times with 1.5 ml of distilled water. Each disk was removed from its hole and dried overnight between sheets of paper towel without exposure with direct refreshment.The disks were examined visually and then analyzed using a Minolta Reflectometer CR-200 calibrated with a standard white tile. L values with standard deviation were calculated.

The histograms shown in Figure 4 show that Trip-TG (diluted 1/50 in 50 mM Hepes buffer) removes dirt when contaminated with salad dressing and when soiled with guar pigment in 50 mM Hepes buffer, pH 7.4, and in 50 mM Hepes buffer, pH 7.4, plus 0.15% AATCC HDL.

The figure 5 presents the results obtained in the experiment on the dose dependence of the response of microsamples to Trip-TG. Trip-TG removed contaminants with salad dressing at a concentration of 1 ppm in 0.15% AATCC heavy-duty liquid detergent.

Figure 6 shows that Trip-TG removed contamination with salad dressing in a micro-sample purification experiment in a 0.1% North American standard AATCC-1993 HDD without clarifier. This detergent contained 18% linear alkyl sulfonates, 25% Zeolite A, 18% soda ash, 0.5% sodium silicate, 22% sodium sulfate, 10% humectant, and 6.3% copolymer or other additives.

This experiment demonstrated the activity of transglucosidase in the removal of contaminants in both heavy duty liquid (HDL) and heavy duty solid (HDD) detergents.

Example 4

Analysis of the cleaning activity of transglucosidase using a tergotometer

In studies using a tergotometer, a 6-tank model 7243S tergotometer (U.S. Testing, Co. Inc. Hoboken, N.J.), maintained at 30 ° C, was used. The stirring speed was set at 100 rpm. Cotton samples (5 for each tergotometer tank) obtained from Warwick Equest Limited, Consett, County Durham, England, stained with food circles, were added to 1 liter of 0.15% AATCC HDL detergent with a water hardness of 6 grains per gallon (diluted from stock solution with a water hardness of 15,000 grains per gallon containing 1.735 M calcium chloride and 0.67 M magnesium chloride) and 25 mm Hepes buffer pH 7.4. After a washing cycle for 30 minutes, the samples were washed three times with 1.5 L of cold tap water, unscrewed for 7 minutes in a spin cycle to remove excess water, and dried overnight at room temperature. The percentage of pollution release (% SRI) was calculated using standard methods after analyzing each pollution using an OTDR. Figure 7 shows that transglucosidase substantially cleans jam contamination compared to a non-enzyme control or a control with unrelated protein, bovine serum albumin (BSA-50, Fraction V, Immunoglobulin and Protease Free) obtained from Rockland Immunochemicals, Gilbertsville, PA.

The above examples demonstrate that transglucosidase causes the effective destruction of xanthan gum and removes certain contaminants from samples of cotton fabrics.

All patents and publications mentioned in the description are indicators of the level reached by experts in the field of technology to which this invention relates. All patents and publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the indicated ends and advantages, as well as those that are inherent therein. The compositions and methods described herein are representative of preferred embodiments and are illustrative and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention described herein as illustrative may suitably be practiced in the absence of any element or elements, limitations or limitations that are not specifically disclosed herein. The terms and expressions that have been used are used as description terms and not limitation, and when using such terms and expressions, it is not intended to exclude any equivalents of the presented and described characteristics or their parts, but it is recognized that various modifications are possible within the scope inventions. Thus, it should be understood that although the present invention is specifically disclosed using preferred embodiments and optional characteristics, those skilled in the art may resort to modifications and variations of the principles disclosed herein, and that such modifications and variations are considered to be within the scope of of the present invention.

In the present description, the invention is described in a broad and general sense. Each of the narrower species and less general groupings falling into the general disclosure also form part of the invention. This includes a general description of the invention with a reservation or negative restriction that removes any item of discussion from the series, regardless of whether the excluded item is specifically indicated in this document.

Claims (19)

1. The use of a composition comprising the transglucosidase enzyme for the decomposition of a natural gum polysaccharide, said natural gum polysaccharide being a substrate for said transglucosidase enzyme. 2. The use according to claim 1, where the specified polysaccharide natural gum is xanthan gum. 3. The use according to claim 1, where the specified transglucosidase enzyme has an activity defined as EC 2.4.1.24 in accordance with the IUBMB nomenclature. 4. The use according to claim 1, wherein said transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to Aspergillus transglucosidase enzyme. 5. The use according to claim 1, where the specified composition further comprises at least one surfactant. 6. The use according to claim 1, where the specified polysaccharide of natural gum is present in the form of contamination on the subject. 7. The use according to claim 6, where the specified item is a fabric. 8. A method of destroying natural gum, comprising contacting the transglucosidase enzyme with a natural gum polysaccharide to destroy said natural gum polysaccharide. 9. The method of claim 8, wherein said transglucosidase enzyme has an activity defined as EC 2.4.1.24 in accordance with the IUBMB nomenclature. 10. The method of claim 8, wherein said natural gum polysaccharide is xanthan gum. 11. The cleaning method, including:
a) contacting an object contaminated with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and
b) maintaining the specified subject and the cleaning composition under conditions sufficient to effectively destroy the specified polysaccharide of natural gum and thereby cleaning the specified subject. 12. The cleaning method according to claim 11, where the specified item is contaminated with food that contains the specified polysaccharide natural gum. 13. The cleaning method according to claim 11, where the specified polysaccharide natural gum includes xanthan gum. 14. The cleaning method according to claim 11, where the specified item is selected from fabrics and hard surfaces. 15. The purification method according to claim 11, wherein said transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to Aspergillus transglucosidase enzyme. 16. The cleaning method according to claim 11, where the specified cleaning composition further comprises at least one surfactant. 17. The cleaning method according to claim 11, wherein said cleaning composition further comprises at least one enzyme selected from proteases, amylases, cellulases, lipases, kutinases, mannanases, pectinases, pectatliases and oxidoreductases to destroy other contaminants. 18. The cleaning method according to claim 11, where the specified cleaning composition has a pH from about 5.0 to about 11.5. 19. The cleaning method of claim 11, wherein said cleaning composition comprises said enzyme at a concentration of from about 0.01 to about 100 million ^-1.

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